Helen M. Tang¹, Catriona L. Halliday¹, Kerri Basile¹, Sharon C.-A. Chen^{1, 2, 3}

¹Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, Westmead, NSW 2145, Australia; ²Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW 2006, Australia; ³Sydney Infectious Diseases Institute, The University of Sydney, Westmead, NSW 2145, Australia

Mucormycosis is a life-threatening infection associated with high morbidity and mortality. Conventional diagnostic methods are limited by low sensitivity and prolonged turnaround times, while commercial polymerase chain reaction (PCR) assays are costly and lack genus/species-specific targets. We developed a novel molecular diagnostic workflow to facilitate the rapid detection of Mucorales directly from clinical specimens. This workflow integrates two in-house in vitro diagnostic (IVD) PCR assays: a real-time Pan-Mucorales PCR, followed by a real-time, multiplex genus/species-specific PCR targeting Rhizopus arrhizus, Rhizopus microsporus, and Mucor spp. Specificity was validated using culture isolates of Mucorales, non-Mucorales fungi, and bacteria. 165 clinical specimens (69 Mucorales-positive and 96 negative), confirmed by an in-house panfungal PCR and DNA sequencing, were used to evaluate the PCR assays. The Pan-Mucorales PCR demonstrated 98.6% sensitivity and 100% specificity, while the multiplex genus/species-specific PCR assay achieved sensitivities of 93.8% for R. arrhizus, 70.8% for R. microsporus, and 75% for Mucor spp., each with 100% specificity. Concordance with the panfungal PCR and sequencing protocol was high, supporting the robustness of the workflow. This novel diagnostic approach has the potential to significantly reduce turnaround times, labour, and costs, aiming to streamline the diagnostic process and improve patient outcomes through timely, precise diagnostics.